Phrenic Nerve Amplitude Research Articles

Inactivity-induced phrenic motor facilitation requires PKCζ activity within phrenic motor neurons.

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF.NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.

Impact of diabetes mellitus on the respiratory function of amyotrophic lateral sclerosis patients.

Respiratory insufficiency and its complications are the main cause of death in amyotrophic lateral sclerosis (ALS). The impact of diabetes mellitus (DM) on respiratory function of ALS patients is uncertain. A retrospective cohort study was carried out. From the 1710 patients with motor neuron disease followed in our unit, ALS and progressive muscular atrophy patients were included. We recorded demographic characteristics, functional ALS rating scale (Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised [ALSFRS-R]) and its subscores at first visit, respiratory function tests, arterial blood gases, phrenic nerve amplitude (PhrenAmpl), and mean nocturnal oxygen saturation (SpO2 mean). We excluded patients with other relevant diseases. Two subgroups were analysed: DIAB (patients with DM) and noDIAB (patients without DM). Independent t-test, χ2 , or Fisher exact test was applied. Binomial logistic regression analyses assessed DM effects. Kaplan-Meier analysis assessed survival. p < 0.05 was considered significant. We included 1639 patients (922 men, mean onset age = 62.5 ± 12.6 years, mean disease duration = 18.1 ± 22.0 months). Mean survival was 43.3 ± 40.7 months. More men had DM (p = 0.021). Disease duration was similar between groups (p = 0.063). Time to noninvasive ventilation (NIV) was shorter in DIAB (p = 0.004); total survival was similar. No differences were seen for ALSFRS-R or its decay rate. At entry, DIAB patients were older (p < 0.001), with lower forced vital capacity (p = 0.001), arterial oxygen pressure (p = 0.01), PhrenAmpl (p < 0.001), and SpO2 mean (p = 0.014). ALS patients with DM had increased risk of respiratory impairment and should be closely monitored. Early NIV allowed for similar survival rate between groups.

Respiratory phenotypes in ALS as determined by respiratory questions of the ALSFRS-R and their relation to respiratory tests.

Respiratory insufficiency and its complications are the main cause of death in amyotrophic lateral sclerosis (ALS). Respiratory symptoms are scored in questions Q10 (dyspnoea) and Q11 (orthopnoea) of the revised ALS functional rating scale (ALSFRS-R). The association of respiratory test alterations with respiratory symptoms is unclear. Patients with ALS and progressive muscular atrophy were included. We recorded retrospectively demographic data, ALSFRS-R, forced vital capacity (FVC), maximal inspiratory (MIP) and expiratory (MEP) pressures, mouth occlusion pressure at 100ms (P0.1), nocturnal oximetry (SpO2 mean), arterial blood gases, phrenic nerve amplitude (PhrenAmpl). Three groups were categorized: G1- normal Q10 and Q11; G2- abnormal Q10; G3- abnormal Q10 and Q11 or only abnormal Q11. A binary logistic regression model explored independent predictors. We included 276 patients (153 men, onset-age 62.6±11.0yrs, disease duration 13.0±9.6mo, spinal-onset 182) with mean survival 40.1±26.0mo. Gender, onset-region and disease duration were similar in G1 (n=149), G2 (n=78) and G3 (n=49). Time to NIV was shorter in G3 (p<0.001), but survival was similar. ALSFRS-R subscores were significantly different (G1>G2>G3, p<0.001), except for lower limb subscore (p=0.077). G2 and G3 patients were older than G1 (p<0.001), had lower FVC, MIP, MEP, PhrenAmpl and SpO2 mean. Independent predictors for G2 were MIP and SpO2 mean; for G3 was only PhrenAmpl. These three distinct ALS phenotypic respiratory categories represent progressive stages of ventilatory dysfunction, supporting ALSFRS-R clinical relevance. Orthopnea is a severe symptom that should prompt NIV, being phrenic nerve response an independent predictor. Early NIV promotes similar survival for G2 and G3.

The long-term impact of ovariectomy on ventilation and expression of phrenic long-term facilitation in female rats.

New Findings What is the central question of this study? Would ovariectomy cause prolonged changes in ventilation and sustained loss of acute, intermittent hypoxia‐induced neuroplasticity or would these outcomes be restored with time? What is the main finding and its importance? Our main findings demonstrate that ovariectomy elicits minimal alteration in overall breathing function but impairs acute, intermittent hypoxia‐induced plasticity for ≤ 12 weeks. Sex hormones are necessary to enable respiratory neuroplasticity, including phrenic long‐term facilitation (pLTF), a form of respiratory motor plasticity elicited by acute, intermittent hypoxia (AIH). Female rats exhibit a progressive increase in phrenic nerve amplitude after AIH characteristic of pLTF only during pro‐oestrus, the stage of the oestrous cycle notable for elevated circulating oestradiol levels. Removal of the ovaries [ovariectomy (OVX)], the primary source of circulating oestradiol, also eliminates AIH‐induced pLTF after 1 week. Ovariectomy is used routinely as a model to examine the impact of sex hormones on CNS structure and function, but the long‐term impact of OVX is rarely examined. Extra‐ovarian sites of oestradiol synthesis, including multiple CNS sites, have been identified and might possess the capacity to restore oestradiol levels, in part, over time, impacting respiratory function and the expression of respiratory neuroplasticity. We examined both ventilation in awake, freely behaving female rats, using barometric plethysmography, and the expression of AIH‐induced pLTF in anaesthetized, ventilated female rats 2 and 12 weeks after OVX and compared them with age‐matched ovarian‐intact female rats. Our findings indicate that chronic OVX had little impact on baseline breathing or in the response to respiratory challenge (10% O2, 5% CO2, balance N2) during plethysmography. However, OVX rats at both 2 and 12 weeks demonstrated a persistent loss of AIH‐induced pLTF relative to control animals (P < 0.01), suggesting that other sources of oestradiol synthesis were insufficient to restore pLTF. These data are consistent with our previous work indicating that oestradiol plays a key role in expression of AIH‐induced respiratory neuroplasticity.

The Long‐Term Impact of Ovariectomy on Ventilation and Expression of Phrenic Long‐Term Facilitation in Female Rats

Estrogen is necessary for the expression of phrenic long-term facilitation (pLTF) in female rats. pLTF, a well-studied model of respiratory neuroplasticity, is characterized by a progressive increase in phrenic nerve amplitude following acute intermittent hypoxia (AIH). Gonadally-intact, adult female rats only develop pLTF in stages of the estrous cycle with high levels of circulating estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating estrogen in females, also prevents AIH-induced pLTF and estrogen supplementation is sufficient to restore pLTF in OVX rats, supporting a key role for estrogen in the development of pLTF. Respiratory neuroplasticity studies using OVX female rats are generally completed ~7-10 days post-OVX; a timeframe sufficient for surgery recovery and for circulating estrogen levels to significantly decline. However, few studies have explored the long-term impact of OVX on respiratory function or the development of respiratory neuroplasticity. Here, we examined ventilation in awake, freely behaving rats and the development of AIH-induced pLTF in adult female rats 12 weeks-post OVX (or sham control surgeries) and compared them to female rats 2 weeks-post OVX. Our hypothesis was that chronic OVX would have minimal long-term impact on ventilation but would continue to impair development of AIH-induced pLTF. Whole body plethysmography was used to measure baseline breathing (20.9% O2, N2 balance) and ventilatory responses to strong chemoreceptor stimulation (10% O2, 5% CO2, N2 balance). Respiratory measures including frequency, tidal volume, minute ventilation, and respiratory neural drive were measured and compared between groups. Following plethysmography, phrenic nerve recordings were used to quantify pLTF. Results indicate that chronic OVX had little impact on baseline breathing function and rats in all experimental groups responded appropriately to respiratory challenge during plethysmography. However, 12 week OVX rats showed a significant increase in tidal volume relative to baseline during respiratory challenge (p≤0.001), different from other groups. This increase in tidal volume production was also reflected in the calculation of tidal volume/inspiratory time, a measure of relative respiratory neural drive (p≤0.01) but not in minute ventilation. OVX had sustained effects on development of respiratory plasticity, as only rats in the 12 week sham control group displayed pLTF, indicated by significantly increased phrenic nerve amplitude 60-min following AIH (p≤0.001). This increase in amplitude was higher than both 12 week and 2 week OVX groups (p≤0.01). ELISA assays also showed a significant correlation between circulating estrogen levels and magnitude of pLTF (p= 0.039) In summary, OVX appears to limit the expression of respiratory neuroplasticity in female rats for up to 12 weeks. Since no significant changes in circulating estrogen are observed in OVX rats over that time, these data are consistent with our prior work indicating that estrogen plays a key role in development of respiratory neuroplasticity.

Low concentrations of nicotine increase frequency and decrease amplitude of respiratory bursts recorded from the hypoglossal nerve, with no change in amplitude of bursts recorded from the phrenic nerve, in the neonatal rat brainstem spinal cord preparation

Nicotinic acetylcholine receptors (nAChRs) are expressed throughout the central respiratory network, including postsynaptically on pattern generating neurons and motor neurons, and presynaptically on both excitatory and inhibitory inputs to these neurons. Normally, specific populations of nAChRs are briefly activated by the endogenous neurotransmitter acetylcholine, which is then rapidly hydrolyzed by acetylcholinesterase. However, with active or passive nicotine exposure, low circulating concentrations of nicotine results in global and prolonged activation of nAChRs, the effects of which are largely unknown. Here, we used the brainstem spinal cord preparation and rhythmic brainstem slice from nicotine‐naïve neonatal rats on postnatal days 1–4 to study the effects of prolonged, global nAChR activation on respiratory motor output. In the brainstem spinal cord preparation, bath application of 250 nM nicotine for 30 minutes resulted in an increase in respiratory burst frequency (21 % above baseline) accompanied by a decrease in hypoglossal nerve amplitude (62 % below baseline), but no change in phrenic nerve amplitude. In the rhythmic brainstem slice, bath application of nicotine concentrations as low as 50 nM caused an increase in frequency (35 % above baseline) and a decrease in amplitude (33 % below baseline) of respiratory bursts recorded from the hypoglossal nerve. These results indicate that low concentrations of nicotine are sufficient to cause changes in the respiratory network, including profound inhibition of hypoglossal motor output.Support or Funding Information5R01HD071302‐07

Development of ventilatory long-term facilitation is dependent on estrous cycle stage in adult female rats

Ventilatory long-term facilitation (vLTF) is a form of respiratory plasticity characterized by a progressive and sustained increase in minute ventilation over time following acute, intermittent hypoxia (AIH). Though vLTF has been repeatedly demonstrated in adult males (rats and humans), few studies have assessed vLTF in adult females and no studies have explored differential expression of vLTF across the normal female estrous cycle. We recently reported that AIH-induced plasticity of phrenic motor output (phrenic long-term facilitation, pLTF), a phenotypically similar form of respiratory plasticity presenting as a sustained increase in phrenic nerve amplitude, develops in adult female rats only during the proestrus stage of the estrous cycle, notable for high levels of serum estrogen. Here, we tested the hypothesis that AIH-induced vLTF would also be estrous-stage dependent; developing in female rats during proestrus, but not estrus. Barometric plethysmography in adult (4–5 months), normally cycling female rats revealed a progressive increase in minute ventilation for 60 min following AIH (5 × 5 min episodes; 10% O2) during proestrus indicative of vLTF, while estrus rats showed no changes in minute ventilation over the same time period. The development of vLTF in proestrus rats was driven by changes in tidal volume production versus respiratory frequency consistent with prior studies. These data are the first to investigate differential vLTF expression across the estrous cycle in adult female rats and highlight the importance of female estrous cycle stage as a critical physiological variable to consider in studies of AIH-induced plasticity.

Difference between the Right and Left Phrenic Nerve Conduction Times, Latency, and Amplitude.

We studied phrenic nerve conduction times in 90 phrenic nerves of 45 normal subjects. The phrenic nerve was stimulated at the posterior border of the sternomastoid muscle in the supraclavicular fossa, just above the clavicle, with bipolar surface electrodes. For recording, positive and negative electrodes were placed on the xiphoid process and at the eighth intercostal bone-cartilage transition, respectively. We studied both the right and left sides to determine whether there was any difference between the two sides. The mean onset latency (± SD) of the right compound muscle action potentials (CMAPs) (5.99±0.39 msec) was significantly shorter than that of the left CMAPs (6.45±0.50 msec). The mean peak latency was significantly shorter in the right CMAPs (10.22±1.33 msec) than the left CMAPs (12.48±2.02 msec). The mean (± SD) amplitude was significantly lower in the left CMAPs (0.42±0.11 mV) than the right CMAPs (0.49±0.10 mV). The difference between the length of the nerve on the right and left sides might have affected the difference in latency between the two sides.

Probing the function of glycinergic neurons in the mouse respiratory network using optogenetics

Glycine is a primary inhibitory transmitter in the ventral medullary respiratory network, but the functional role of glycinergic neurons for breathing remains a matter of debate. We applied optogenetics to selectively modulate glycinergic neuron activity within regions of the rostral ventral respiratory column (VRC). Responses of the phrenic nerve activity to the light-driven stimulation were studied in the working heart-brainstem preparation from adult glycine transporter 2 Cre mice (GlyT2-Cre), which received a unilateral injection of a Cre-dependent AAV virus into Bötzinger and preBötzinger Complex. Sustained light stimulation from the ventral medullary surface resulted in a substantial depression of the phrenic nerve (PN) frequency, which in most cases was compensated by an increase in PN amplitude. Periodic, burst stimulation with variable intervals could alter and reset respiratory rhythm. We conclude that unilateral activation of the rostral VRC glycinergic neurons can significantly affect respiratory pattern by lengthening the expiratory interval and modulating phase transition.

T39. Diaphragm motor responses to phrenic nerve stimulation: Surface and needle recordings

In studies of phrenic nerve (PN) conduction in amyotrophic lateral sclerosis (ALS) both motor response amplitude and latency have been reported as abnormal. However, correlation with diaphragm motor unit loss, and with diaphragmatic function has not been fully evaluated. We studied 83 patients with ALS, and 21 patients referred with clinically suspected phrenic nerve lesions whose studies were normal. PN responses elicited by percutaneous electrical stimulation in the neck were recorded using superficial electrodes placed at the surface markings of the diaphragm on the chest wall, and a concentric needle electrode inserted into the diaphragmatic costal fibres. Electromyography of diaphragm was performed to analyse motor unit morphology and recruitment. Diaphragm changes were scored as: minor, no fibrillation/sharp-waves (fibs-sw) and mild loss of motor units on activation; moderate, fibs-sw and moderate loss of motor units on activation; severe, fibs-sw and few remaining complex motor units with very increased firing-rate. The 21 controls and 83 ALS patients were matched for age. In controls, the only significant correlation between surface and needle recording was for negative-peak amplitude (p = 0.03). In ALS patients, amplitudes and negative-peak area were highly correlated (p < 0.001), as were PN motor latencies (p = 0.002). Forced vital capacity (FVC) was highly correlated with both amplitude (p < 0.001) and PN latency (p < 0.02), whichever electrode was used. PN amplitude recording with needle electrode was consistent with EMG findings in the diaphragm. In general, patients with peak-to-peak amplitude <1.0 mV (20% percentile of the median value) on needle recording tended to show severe changes on needle EMG of the diaphragm, and those with amplitude ⩾1.0 mV to <1.5 mV (1st IQR of the median value) had moderate changes in diaphragm EMG. Generally, EMG changes in internal intercostal muscles mirrored findings observed in diaphragm. In ALS, PN motor amplitude/area and latency measurements recorded by surface electrodes are highly correlated with needle EMG findings in the diaphragm. CMAP amplitude/area measurements showed high correlation with FVC. In ALS, amplitude/area of the motor PN response, recorded by surface or needle electrodes, correlates with dysfunction of the diaphragm.

Phrenic Nerve Conduction Study in the Early Stage of Guillain-Barre Syndrome as a Predictor of Respiratory Failure.

Background:Guillain-Barré syndrome (GBS) has unpredictable clinical course with severe complication of respiratory failure.Objective:To identify clinical profiles and electrophysiological study particularly non-invasive Phrenic nerve conduction study in patients of early GBS to predict respiratory failure.Methods:64 adult (age≥18yrs) patients of early GBS (onset ≤ 14 days) during the study period from January 2014 to October 2015 were evaluated by clinical profiles of age, gender, antecedent infection, time to peak disability, single breath counts, cranial nerve involvement, autonomic dysfunction and non-invasive Phrenic nerve conduction study. Patients with predisposition factors of polyneuropathy like diabetes mellitus, hypothyroidism, vitamin deficiency, renal failure were excluded.Results:Among 64 patients abnormal phrenic nerve conduction study was seen in 65.62% cases (42/64) and 45.23% (19/42) of them developed respiratory failure. Phrenic nerve sum latency, amplitude, duration and area were abnormal in those who developed respiratory failure and they had sum of phrenic nerve latency >28 msec, sum of CMAP amplitude <300 μV, sum of CMAP duration >50 msec and sum of area < 4 mVmS. None with normal phrenic nerve study developed respiratory failure. It was found that age, gender, preceding infection, autonomic involvement and types of GB syndrome had no influence on development of respiratory failure (p>0.05). Rapid disease progression to peak disability, more severe disease, shorter single breath counts and cranial nerve involvement were seen more often in patients with respiratory failure.Conclusion:Abnormal Phrenic nerve conduction study in the early Guillain-Barré syndrome might be of great value independently in predicting impending respiratory failure.

Diaphragm motor responses to phrenic nerve stimulation in ALS: Surface and needle recordings

ObjectiveIn studies of phrenic nerve (PN) conduction in amyotrophic lateral sclerosis (ALS) both motor response amplitude and latency have been reported as abnormal. However, correlation with diaphragm motor unit loss, and with diaphragmatic function has not been fully evaluated. MethodsWe studied 83 patients with ALS, and 21 patients referred with clinically suspected phrenic nerve lesions whose studies were normal. PN responses elicited by percutaneous electrical stimulation in the neck were recorded using superficial electrodes placed at the surface markings of the diaphragm on the chest wall, and a concentric needle electrode inserted into the diaphragmatic costal fibres. Electromyography of diaphragm was performed to analyse motor unit morphology and recruitment. ResultsThe 21 controls and 83 ALS patients were matched for age. In controls, the only significant correlation between surface and needle recording was for negative-peak amplitude (p = 0.03). In ALS patients, amplitudes and negative-peak area were highly correlated (p < 0.001), as were PN motor latencies (p = 0.002). Forced vital capacity (FVC) was highly correlated with both amplitude (p < 0.001) and PN latency (p < 0.02), whichever electrode was used. PN amplitude recording with needle electrode was consistent with EMG findings in the diaphragm. ConclusionIn ALS, PN motor amplitude/area and latency measurements recorded by surface electrodes are highly correlated with needle EMG findings in the diaphragm. CMAP amplitude/area measurements showed high correlation with FVC. SignificanceIn ALS, amplitude/area of the motor PN response, recorded by surface or needle electrodes, correlates with dysfunction of the diaphragm.

Peripheral neuropathy in stable chronic obstructive pulmonary disease: Is the phrenic nerve more affected compared with other peripheral nerves?

Background Chronic obstructive pulmonary disease (COPD) is a multicomponent disease with extrapulmonary manifestation. Peripheral nerve affection may be one of the results or mechanism of COPD. Objective The aim of this study was to evaluate peripheral neuropathy in patients with stable COPD and whether these changes are more or less marked in the phrenic nerve when compared with other peripheral nerves. Patients and methods Eighty patients with COPD and 60 healthy individuals as a control group were included. All were subjected to the following: (i) full history taking; (ii) clinical and neurological examination; (iii) chest radiography; (iv) high-resol ution computed tomography scanning of the chest; (v) arterial blood gases analysis at room air; and (vi) spirometric tests. Results There was a statistically significant increase in smoking packs/year, respiratory rate, hemoglobin, and white blood cells in the COPD group. There was a significant decrease in oxygen saturation, pH, arterial oxygen tension, forced expiratory volume in 1 s, forced expiratory volume in 1 s/forced vital capacity, and forced vital capacity% in the study group when compared with the control group, whereas there was a significant increase in CO2 tension in the COPD group. There was a significant increase in the distal latency of all nerves and a significant decrease in the amplitude and conduction velocity of all nerves, except the amplitude of the phrenic nerve in the COPD group. The difference for phrenic nerve amplitude was statistically nonsignificant. In the COPD group, abnormal motor activity in the median, ulnar, common peroneal, and phrenic nerves of 30, 22.5, 35, and 22.5%, respectively, was reported, whereas abnormal sensory activity in the median, ulnar, and sural nerves of 30.0, 27.5, and 42.5%, respectively, was reported. Finally, we found a statistically significant increase in nerve abnormalities with increased severity of the disease. Conclusion COPD had a significant affection on peripheral nerves, either motor or sensory. In addition, COPD had a significant affection on the phrenic nerve and muscle of the diaphragm. However, this affection of the phrenic nerve was confined to grades III and IV and was the least affection of studied nerves.

Phrenic nerve deficits and neurological immunopathology associated with acute West Nile virus infection in mice and hamsters

Neurological respiratory deficits are serious outcomes of West Nile virus (WNV) disease. WNV patients requiring intubation have a poor prognosis. We previously reported that WNV-infected rodents also appear to have respiratory deficits when assessed by whole-body plethysmography and diaphragmatic electromyography. The purpose of this study was to determine if the nature of the respiratory deficits in WNV-infected rodents is neurological and if deficits are due to a disorder of brainstem respiratory centers, cervical spinal cord (CSC) phrenic motor neuron (PMN) circuitry, or both. We recorded phrenic nerve (PN) activity and found that in WNV-infected mice, PN amplitude is reduced, corroborating a neurological basis for respiratory deficits. These results were associated with a reduction in CSC motor neuron number. We found no dramatic deficits, however, in brainstem-mediated breathing rhythm generation or responses to hypercapnia. PN frequency and pattern parameters were normal, and all PN parameters changed appropriately upon a CO2 challenge. Histological analysis revealed generalized microglia activation, astrocyte reactivity, T cell and neutrophil infiltration, and mild histopathologic lesions in both the brainstem and CSC, but none of these were tightly correlated with PN function. Similar results in PN activity, brainstem function, motor neuron number, and histopathology were seen in WNV-infected hamsters, except that histopathologic lesions were more severe. Taken together, the results suggest that respiratory deficits in acute WNV infection are primarily due to a lower motor neuron disorder affecting PMNs and the PN rather than a brainstem disorder. Future efforts should focus on markers of neuronal dysfunction, axonal degeneration, and myelination.

Maternal Protein Restriction Increases Respiratory and Sympathetic Activities and Sensitizes Peripheral Chemoreflex in Male Rat Offspring1–3

Background: Maternal protein restriction in rats increases the risk of adult offspring arterial hypertension through unknown mechanisms.Objectives: The aims of the study were to evaluate the effects of a low-protein (LP) diet during pregnancy and lactation on baseline sympathetic and respiratory activities and peripheral chemoreflex sensitivity in the rat offspring.Methods: Wistar rat dams were fed a control [normal-protein (NP); 17% protein] or an LP (8% protein) diet during pregnancy and lactation, and their male offspring were studied at 30 d of age. Direct measurements of baseline arterial blood pressure (ABP), heart rate (HR), and respiratory frequency (Rf) as well as peripheral chemoreflex activation (potassium cyanide: 0.04%) were recorded in pups while they were awake. In addition, recordings of the phrenic nerve (PN) and thoracic sympathetic nerve (tSN) activities were obtained from the in situ preparations. Hypoxia-inducible factor 1α (HIF-1α) expression was also evaluated in carotid bifurcation through a Western blotting assay.Results: At 30 d of age, unanesthetized LP rats exhibited enhanced resting Rf (P = 0.001) and similar ABP and HR compared with the NP rats. Despite their similar baseline ABP values, LP rats exhibited augmented low-frequency variability (∼91%;P = 0.01). In addition, the unanesthetized LP rats showed enhanced pressor (P = 0.01) and tachypnoeic (P = 0.03) responses to peripheral chemoreflex activation. The LP rats displayed elevated baseline tSN activity (∼86%;P = 0.02) and PN burst frequency (45%;P = 0.01) and amplitude (53%;P = 0.001) as well as augmented sympathetic (P = 0.01) and phrenic (P = 0.04) excitatory responses to peripheral chemoreflex activation compared with the NP group. Furthermore, LP rats showed an increase of ∼100% in HIF-1α protein density in carotid bifurcation compared with NP rats.Conclusion: Sympathetic-respiratory overactivity and amplified peripheral chemoreceptor responses, potentially through HIF-1α–dependent mechanisms, precede the onset of hypertension in juvenile rats exposed to protein undernutrition during gestation and lactation.

Inactivity‐induced phrenic motor facilitation does not require new protein synthesis (713.8)

Reduced respiratory neural activity elicits a TNFα‐ and atypical PKC‐dependent form of spinal plasticity in phrenic motor output termed inactivity‐induced phrenic motor facilitation (iPMF), a long‐lasting increase in phrenic nerve burst amplitude apparent once respiratory neural activity is restored. Many forms of neuroplasticity require protein synthesis for long‐term expression, including the most widely studied form of spinal respiratory plasticity, phrenic long‐term facilitation (pLTF; Baker‐Herman and Mitchell, 2002) elicited following acute intermittent hypoxia. We hypothesized that iPMF would also require de novo protein synthesis. Briefly, Sprague Dawley rats were urethane anesthetized, vagotomized, paralyzed, and prepared for electrophysiological recording. Rats received intrathecal injection (C4) of vehicle (aCSF; n=5) or the protein synthesis inhibitor emetine (n=6; 1ug/kg; Baker‐Herman and Mitchell, 2002) ~15 min prior to a prolonged (30min) period of neural apnea. As expected, in aCSF treated rats, phrenic nerve amplitude was significantly increased compared to baseline 5 min following reversal of neural apnea (53±11, %baseline) and was maintained for &gt;60 min (54±16, %baseline), indicating long‐lasting iPMF. Contrary to our hypothesis, rats receiving emetine also expressed long‐lasting iPMF such that phrenic nerve burst amplitude was significantly increased from baseline at both 5 (41±8, %baseline) and 60 min (42±12,%baseline) following neural apnea (p&lt;0.05). Importantly, phrenic nerve burst amplitude in rats receiving intrathecal emetine was not significantly different from rats receiving aCSF at any time point following neural apnea (p&gt;0.05). Together, these data suggest that iPMF is a form of long‐lasting plasticity that does not require new protein synthesis.Grant Funding Source: Supported by NIH HL105511

Respiratory, metabolic and cardiac functions are altered by disinhibition of subregions of the medial prefrontal cortex

The prefrontal cortex (PFC) is referred to as the visceral motor cortex; however, little is known about whether this region influences respiratory or metabolic outflows. The aim of this study was to describe simultaneous changes in respiratory, metabolic and cardiovascular functions evoked by disinhibition of the medial PFC (mPFC) and adjacent lateral septal nucleus (LSN). In urethane-anaesthetized rats, bicuculline methiodide was microinjected (2 mm; GABA-A receptor antagonist) into 90 sites in the mPFC at 0.72-4.00 mm from bregma. Phrenic nerve amplitude and frequency, arterial pressure, heart rate, splanchnic and lumbar sympathetic nerve activities (SNA), expired CO2, and core and brown adipose tissue temperatures were measured. Novel findings included disturbances to respiratory rhythm evoked from all subregions of the mPFC. Injections into the cingulate cortex evoked reductions in central respiratory function exclusively, whereas in ventral sites, particularly the infralimbic region, increases in respiratory drive and frequency, and metabolic and cardiac outflows were evoked. Disinhibition of sites in surrounding regions revealed that the LSN could evoke cardiovascular changes accompanied by distinct oscillations in SNA, as well as increases in respiratory amplitude. We show that activation of neurons within the mPFC and LSN influence respiratory, metabolic and cardiac outflows in a site-dependent manner. This study has implications with respect to the altered PFC neuronal activity seen in stress-related and mental health disorders, and suggests how basic physiological systems may be affected.

Nucleus tractus solitarii (nTS) is required for phrenic long term facilitation (pLTF) after acute intermittent hypoxia (AIH)

Repeated episodes of acute hypoxia lead to pLTF which is thought to involve local mechanisms in the spinal cord. However, carotid body denervation decreases pLTF indicating chemoreceptor afferent input also contributes to pLTF. We hypothesized that the nTS, the first integration site of chemoreceptor afferents, is critical to the generation and maintenance of pLTF. Anesthetized, vagotomized, paralyzed and ventilated male rats were exposed to 10 hypoxic episodes (10% O2, 45sec, 5 min intervals) following bilateral nTS microinjection of aCSF or the GABA receptor agonist muscimol to inhibit nTS activity. After aCSF injection (n=4) integrated phrenic nerve activity increased during hypoxia (567±222% baseline) and amplitude remained elevated ≥1hr after AIH (131±28% baseline). Muscimol injection (2mM; 60nL) prior to AIH blocked increases in phrenic activity during hypoxia (7±14% baseline; n=3) and no LTF occurred (−3±8% baseline). Furthermore, muscimol injections after pLTF induction (130±33% baseline; n=3) decreased phrenic nerve amplitude toward pre‐AIH levels (6±10% baseline; n=3). Results indicate that nTS neuronal activity is required for the development and maintenance of pLTF. HL98602, AvH 1135664

Neuromedin U causes biphasic cardiovascular effects and impairs baroreflex function in rostral ventrolateral medulla of spontaneously hypertensive rat

Neuromedin U (NMU) causes biphasic cardiovascular and sympathetic responses and attenuates adaptive reflexes in the rostral ventrolateral medulla (RVLM) and spinal cord in normotensive animal. However, the role of NMU in the pathogenesis of hypertension is unknown. The effect of NMU on baseline cardiorespiratory variables in the RVLM and spinal cord were investigated in urethane-anaesthetized, vagotomized and artificially ventilated male spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats (WKY). Experiments were also conducted to determine the effects of NMU on somatosympathetic and baroreceptor reflexes in the RVLM of SHR and WKY. NMU injected into the RVLM and spinal cord elicited biphasic response, a brief pressor and sympathoexcitatory response followed by a prolonged depressor and sympathoinhibitory response in both hypertensive and normotensive rat models. The pressor, sympathoexcitatory and sympathoinhibitory responses evoked by NMU were exaggerated in SHR. Phrenic nerve amplitude was also increased following intrathecal or microinjection of NMU into the RVLM of both strains. NMU injection into the RVLM attenuated the somatosympathetic reflex in both SHR and WKY. Baroreflex sensitivity was impaired in SHR at baseline and further impaired following NMU injection into the RVLM. NMU did not affect baroreflex activity in WKY. The present study provides functional evidence that NMU can have an important effect on the cardiovascular and reflex responses that are integrated in the RVLM and spinal cord. A role for NMU in the development and maintenance of essential hypertension remains to be determined.

Increasing brain serotonin corrects CO2 chemosensitivity in methyl‐CpG‐binding protein 2 (Mecp2)‐deficient mice

Mice deficient in the transcription factor methyl-CpG-binding protein 2 (Mecp2), a mouse model of Rett syndrome, display reduced CO2 chemosensitivity, which may contribute to their breathing abnormalities. In addition, patients with Rett syndrome and male mice that are null for Mecp2 show reduced levels of brain serotonin (5-HT). Serotonin is known to play a role in central chemosensitivity, and we hypothesized that increasing the availability of 5-HT in this mouse model would improve their respiratory response to CO2. Here we determined the apnoeic threshold in heterozygous Mecp2-deficient female mice and examined the effects of blocking 5-HT reuptake on the CO2 response in Mecp2-null male mice. Studies were performed in B6.129P2(C)-Mecp2(τm1.1Bird) null males and heterozygous females. In an in situ preparation, seven of eight Mecp2-deficient heterozygous females showed arrest of phrenic nerve activity when arterial CO2 was lowered to 3%, whereas the wild-types maintained phrenic nerve amplitude at 53 ± 3% of maximal. In vivo plethysmography studies were used to determine CO2 chemosensitivity in null males. These mice were exposed sequentially to 1, 3 and 5% CO2. The percentage increase in minute ventilation in response to increased inspired CO2 was less in Mecp2(-/y) than in Mecp2(+/y) mice. Pretreatment with citalopram, a selective 5-HT reuptake inhibitor (2.5 mg kg(-1) i.p.), 40 min prior to CO2 exposure, in Mecp2(-/y) mice resulted in an improvement in CO2 chemosensitivity to wild-type levels. These results suggest that decreased 5-HT in Mecp2-deficient mice reduces CO2 chemosensitivity, and restoring 5-HT levels can reverse this effect.